In 1966, Ishizaka established that the human allergen reaginic antibodies belong to a distinct class of immunoglobulins, IgE. There followed a decade of remarkable sequence of events. Sensitization of the human and monkey skin to Prausnitz-Kunster reaction by IgE antibodies was demonstrated. It was recognized that the role of IgE is central to the release mechanisms of chemical mediators of anaphylaxis; it mediates immunologic release of histamine from human leucocytes; it primes the human lung tissue for antigen-induced release of histamine and Slow Reacting Substance of Anaphylaxis; it triggers the release of eosinophil chemotactic factor from human lung; and its avidity for attachment, through its Fc portion, to the receptors on the surface of mast cells and basophil granules was shown. It has been inferred that the number and the affinity of IgE antibodies bound to the basophil granulocytes determines the sensitivity of this cell to the allergen, while the histamine release induced by the antigen-antibody reaction on the cell surface is the function of the intracellular enzyme system and cyclic AMP level.
Dating back to the early part of the 20th century, patients with hay fever were treated with injections of incriminated allergens, albeit without understanding the pathologic bases of the disease or the pharmacologic bases for the efficacy of the therapy. Johansoon's observation, in 1967, of augmented levels of serum IgE in atopic patients sparked intense interest in this relationship; individuals with inhalant allergies were found to display seasonal peaks in their serum IgE levels; abatement of allergic symptomatology with immunotherapy was documented. Partial suppression of seasonal peaks following specific immunotherapy was demonstrated and the inter-relationship of levels of IgE and IgG in atopic subjects, and the changes induced by specific immunotherapy have been illuminated.
The advent of the enzyme-labelled antibodies was a major event in the progress of immunochemistry. The immunoperoxidase techniques are now well established investigative tools of a high order of sensitivity and specificity. A measure of their versatility and usefulness is reflected in the diversity of their applications. These immunotracing methods have permitted precise cellular and tissue localization of a very large number of specific antigenic substances and antibodies; notable among these: alpha trypsin in liver, carcinoembryonic antigen in colonic carcinoma, Hepatitis B surface antigen, alpha feto-protein, Herpes simplex virus antigen, and several hormones (thyrocalcitonin, pancreatic, and pituitary). The sensitivity of this method has been shown to surpass that of radioimmunoassay. The localization of light chains and immunoglobulins with these procedures has also been extensively investigated in lymphoid and plasma cells, in bullous dermatosis, in Hodgkin's disease, and in renal diseases where the role of immune mechanism has been invoked.
In 1942, the technique of immunofluorescence for the identification of specific antigenic substances and tissues that escape detection by other histochemical methods was described. Immunofluorescence, however, has continued to pose significant technical difficulties including the requirement of "snap-freezing" of the tissue, the poverty of cellular detail with the use of darkfield condenser, and the distressingly short life of fluorescence.
In 1966, Nakane & Pierce published a report demonstrating that peroxidase could be coupled to an antibody by a simple procedure to produce a stable conjugate. The intact immunological reactivity of such a conjugate was shown to render it eminently suitable for use in immunotracing methods, in a fashion similar to that of fluorescein-labelled antibody. Almost simultaneously, the excellent resolution provided by the oxidation product of 3,3'-diaminobenzidine was put to use in conjunction with peroxidase for histochemical localization purposes. The peroxidase-3,3'-diaminobenzidine system was subsequently employed in the "immunoglobulin-enzyme bridge" technique, in which after the interaction of the specific primary antiserum to the antigen of interest, a second antiserum from a different animal species is applied to provide the "bridge" to the antiperoxidase. The IgG molecule is known to carry two binding sites; it has been inferred that one affords localization to the primary antiserum while the second binds the antiperoxidase, which in turn fixes the peroxidase and its chromogenic substrate to the original antigen under study. The enzyme-chromogenic system has not heretofore been used for the detection of IgE.
Accordingly, it is the object of this invention to provide a method by which the enzyme-chromogenic reagent technique can be applied to the detection of allergen-specific IgE antibodies in biologic fluids of atopic individuals and also to provide a test kit for carrying out the procedure. This and other objects of the invention will become apparent to those skilled in this art from the following detailed description.